The long-term goal of this research is to understand the role(s) of gap junctions in blood vessel formation. Insights gained from these studies will enable modulation of adult vessel formation for the treatment of prevalent pathologies, as well as for the generation of tissues ex vivo for regenerative medicine strategies. Gap junction channels can be composed of any of ~20 known connexin (Cx) proteins; however, only Cx37, Cx40, Cx43 and Cx45 are expressed in the vasculature. The overarching hypothesis of our studies is that distinct gap junction channel proteins expressed in the vasculature play unique roles at different stages of blood vessel formation. Our previous studies provide direct evidence that gap junction channels composed of Cx43 form between endothelial cells and mesenchymal cells that they recruit. Furthermore, gap junction channel formation and/or communication is necessary to mediate the activation of TGF- 2, which is required for endothelial-induced mural cell differentiation. In the current grant period, we have determined that gap junction channels composed of Cx45 also mediate this process; whereas, channels composed of Cx40 do not. Furthermore, it appears that specific phosphorylation sites within the cytoplasmic region of Cx43 are required, suggesting that associated intracellular signaling downstream of gap junction channel formation may be necessary for TGF-2 activation. We have further determined that calcium signaling, which is activated upon heterocellular gap junction formation, appears to play a role in endothelial- induced mural cell differentiation. Our proposed studies will further dissect the intracellular signaling pathways downstream of Cx43- and Cx45-composed gap junction channels that form between endothelial and mesenchymal cells and enable endothelial-induced mural cell differentiation. We will also determine the role(s) of Cx37 and Cx40, which do not mediate endothelial-induced mural cell differentiation, in the regulation of endothelial cell differentiation and growth control. Both are highly upregulated in differentiating endothelial cells; whereas, Cx43 and Cx45 are downregulated. Thus, our studies will further dissect and define the specific roles of distinct Cx proteins expressed in the vasculature. PUBLIC HEALTH RELEVANCE: Blood vessels are made up of two cell layers; endothelial cells comprise the inner luminal layer of vessels and mural cells (pericytes and vascular smooth muscle cells) make up the surrounding vessel wall and control blood flow and vessel stability. We are studying how communication between endothelial cells and mural cells helps to promote vessel formation and stability. Insights gained from our developmental studies may help to develop clinical strategies for treating common diseases including atherosclerosis, diabetes, and cancer.